Aspects of the synthesis of an exceptionally preorganized self-immolative spacer-phenolate unit

Article abstract of DOI:10.1055/s-0028-1083296

This study introduces a highly preorganized self-immolative spacer that is coupled to a nonfluorescent leaving group. The water-soluble compound can rapidly liberate a water-insoluble fluorescent precipitate by intramolecular attack of a free amine on a phenolic ester group via a six-membered transition state. One of the crucial steps in the synthesis of this molecule was a sterically very unfavored nucleophilic substitution to create a tertiary ether; this was optimized using model compounds. Another key challenge was deprotection by catalytic hydrogenation at low temperatures in order to furnish the hydroacetate of the free amine without further fragmentation. LC-MS and fluorescence studies showed that this compound collapsed instantaneously in methanol, as well as within a wide pH range in buffered aqueous media. Georg Thieme Verlag Stuttgart · New York.

Full text of DOI:10.1055/s-0028-1083296

318
PAPER
Aspects of the Synthesis of an Exceptionally Preorganized Self-Immolative
Spacer–Phenolate Unit
A
S
elf-Immolative Spa
i
cer–Ph
c
enolate
U
n
h
it ael Waibel, Xiao-Bing Zhang,1 Jens Hasserodt*
Laboratoire de Chimie, UMR CNRS 5182, Université de Lyon-ENS, 46 allée d’Italie, 69364 Lyon cedex 07, France
Fax +33(4)72728860; E-mail: Jens.Hasserodt@ens-lyon.fr
Received 13 September 2008; revised 26 September 2008
In our ongoing research on the design of fluorogenic
Abstract: This study introduces a highly preorganized self-immo-
lative spacer that is coupled to a nonfluorescent leaving group. The
water-soluble compound can rapidly liberate a water-insoluble flu-
orescent precipitate by intramolecular attack of a free amine on a
probes, we were interested in developing an efficient syn-
thesis of molecule 1 that, it was hoped, would rapidly
fragment in aqueous media due to its highly preorganized
phenolic ester group via a six-membered transition state. One of the structure, thus liberating lactam 2 and fluorescent precip-
crucial steps in the synthesis of this molecule was a sterically very
unfavored nucleophilic substitution to create a tertiary ether; this
was optimized using model compounds. Another key challenge was
itate 3 (Scheme 1).
Compound 3, the methoxy derivative of 2-(2-hydroxy-
phenyl)quinazolin-4(3H)-one (HPQ), is rare in its proper-
ties; it is both water-insoluble and highly fluorescent in
the solid state.4 Both its fluorescence and insolubility are
intimately tied to internal hydrogen bonding between the
phenolic hydrogen and the imine nitrogen.5 However, by
masking the phenol group, compounds can be obtained
that are water-soluble and not fluorescent.
deprotection by catalytic hydrogenation at low temperatures in or-
der to furnish the hydroacetate of the free amine without further
fragmentation. LC-MS and fluorescence studies showed that this
compound collapsed instantaneously in methanol, as well as within
a wide pH range in buffered aqueous media.
Key words: self-immolative spacer, preorganization, tertiary ether
formation, low temperature hydrogenation, fluorescence
Central to this project is the strong preorganization of the
spacer unit to guarantee fast, in an ideal case instanta-
neous, fragmentation. One of the two preorganizational
elements is the rotationally restricted N–C bond being part
of a pyrrolidine moiety that is derived from proline. The
second and synthetically more challenging element to
raise preorganization is the quaternary center a to the phe-
nolic ester, which constitutes an exploitation of the
Thorpe–Ingold effect. This geminal dimethyl motive was
also chosen to suppress ester hydrolysis, which is known
to be catalyzed intramolecularly by the imine nitrogen of
HPQ.6
The concept of self-immolative spacers has found wide
application in the domain of prodrug activation2 and in the
development of enzyme assays.3 Agar plate based assays
have proved to be suitable for the high-throughput screen-
ing of large candidate libraries. Probes that yield fluores-
cent precipitates (rather than colorimetric ones) directly
on the agar plate may allow highly sensitive tests to be es-
tablished. Such a format becomes possible if the probe
molecule is water-soluble while the liberated fluorophore
is not, thereby effectively repressing diffusion and thus
signal dilution.
O
N
O
O
H
N
O
N
buffer
pH ≥ 6
+
+
–
NH
O
N
–
H2
OAc
NH
O
O
O
OMe
1⋅HOAc
OMe
O
O
NH
O
N
+
OMe
O
3: fluorescent,
not water-soluble
N
H
2
Scheme 1 Fragmentation of fluorophore-spacer unit 1 in aqueous media, liberating fluorescent precipitate 3
SYNTHESIS 2009, No. 2, pp 0318–0324
x
x
.
x
x
.2
0
0
8
Advanced online publication: 19.12.2008
DOI: 10.1055/s-0028-1083296; Art ID: Z21208SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
A Self-Immolative Spacer–Phenolate Unit
319
O
N
Establishing the quaternary carbon was one of the key
challenges in the synthesis of 1. Although there might be
different strategies for its formation, such as ester enolate
alkylation,7 we envisaged a nucleophilic substitution reac-
tion as shown in Scheme 2.
NH
OMe
R
R
O
R
O
O
OH
7: R = H
8: R = Me
EtO
O
4
O
Figure 1
EtO
N
+
Cbz
6
Subsequently, compound 5 (Scheme 2) was synthesized
by Cbz protection,11 followed by tosylation12 of commer-
cially available (S)-prolinol. Treatment of 4 with sodium
hydride, followed by reaction with 5 gave, not unexpect-
edly, complex product mixtures that were difficult to ana-
lyze. NMR and LC-MS analysis of these mixtures led us
TsO
N
Cbz
5
Scheme 2 Retrosynthetic assembly of tertiary ether 6
While it is obvious that this transformation is sterically to conclude that the carbamate function was involved in
demanding, it has the advantage of furnishing the already side reactions. Therefore, we decided to use 9 as a model
relatively complex ether 6 in only one step from commer- compound, which should make it easier to analyze and op-
cial 4 and readily available 5. The literature only mentions timize this reaction (Scheme 3).
sterically very hindered ether-forming reactions when
OTs
phenolates are used as nucleophiles, for example, in the
O
i) 4, NaH, DMF
synthesis of clofibrate analogues8 or in the formation of
r.t., 30 min
O
benzoxazines.9 However, if the alcohol is aliphatic, and
hence less nucleophilic, literature examples are rare.
Probably the classic knowledge that such reactions are
sterically disfavored has discouraged their inclusion in
synthetic schemes and attempts for optimization have not
been made. In fact, we found only one study where such a
tertiary ether unit is formed in low yield by reaction of a
rather inert alcohol with a tertiary bromide.10 Our ap-
proach represents the opposite sense of attack of a tertiary
alcohol on a primary tosylate.
4
+
EtO
ii) 9, DMF
80 °C, 2 h
91%
9
10
Scheme 3 Model reaction, using 9 to form tertiary ether 10
Compound 4 was thus deprotonated with sodium hydride
and reacted under various conditions with 9, that, in re-
turn, was obtained by tosylation12a of commercial cyclo-
hexylmethanol. The results of this investigation can be
summarized as follows: In tetrahydrofuran, no reaction
took place at all, either by refluxing the mixture, or by ex-
tending the reaction time, or by the use of an excess of so-
dium hydride. In N,N-dimethylformamide, no conversion
was observed at room temperature. However, by raising
the temperature to about 80 °C the transformation became
surprisingly fast, yielding 10 in only two hours in high
yield. To our knowledge, formation of an aliphatic ether
at a quaternary center in high yield has not yet been de-
scribed. Our results demonstrate that in spite of its steric
demand this reaction can be effectively conducted at rea-
sonable temperatures, provided that additional functional
groups present in the molecule are compatible.
The second challenge in this study was the deprotection
and isolation of compound 1 in order to test its cyclization
tendency in aqueous buffered media. In this particular
case, removal of the benzyloxycarbonyl (Cbz) group by
catalytic hydrogenation was not trivial, regarding the high
(in the other context of this project very much desired)
tendency of the free amine to intramolecularly attack the
activated phenol ester. Conditions had to be found that
would allow deprotection, yet would prevent cyclization.
This study describes the synthesis of 1, the optimization of
two interesting key steps, and finally the fragmentation
tendency of the spacer unit at different pH values in buff-
ered aqueous media as followed by LC-MS and fluores-
cence spectroscopy.
The conditions worked out in the preparation of 10 were
then applied to the reaction of 4 with 5 (Scheme 4). As ex-
pected, the yield of 6 was lower than the one for 10, likely
because of involvement of the carbamate function as al-
ready mentioned. Still, using the optimized reaction con-
ditions from above, a rather satisfactory yield of 42% is
obtained. For comparison, a yield of only 15% was
achieved in the only literature example of comparable
steric demand.10
Initially we found that the phenolic ester of model com-
pound 7 is easily hydrolyzed, which is in accord with a
previous report (Figure 1).6 These authors proposed that
this effect is due to anchimeric assistance of the neighbor-
ing imine moiety in HPQ. When we used pivaloylated
HPQ 8 instead, no background hydrolysis could be ob-
served. Apparently, the quaternary center a to the phenol-
ic ester carbonyl prevents access by the imine nitrogen to
the ester carbonyl and, thus, its assistance in hydrolysis.
In the following, compound 6 was hydrolyzed13 to give
quantitatively the corresponding acid 11. Reaction with
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320
M. Waibel et al.
PAPER
O
i) NaH, DMF
r.t., 30 min
i) LiOH, THF–MeOH–H2O
r.t., 12 h
3, EDC,
HOBt, DMF
O
4
6
HO
N
ii) 5, DMF
80 °C, 2 h
42%
ii) HCl
100%
r.t., 12 h
85%
Cbz
11
O
N
O
O
N
O
O
H2, 5% Pd/C
MeOH
O
NH
O
N
H
NH
O
N
2 + 3
Cbz
r.t., 20 min
100%
OMe
1
12
OMe
Scheme 4 Synthesis of 12 and in situ fragmentation via 1 after deprotection in methanol
34,5 using N-(3-dimethylaminopropyl)-N¢-ethylcarbodi- to conduct the hydrogenation at low temperatures. The re-
imide and 1-hydroxy-1H-benzotriazole in N,N-dimethyl- sults of our extensive investigation into this deprotection
formamide as coupling agents14 furnished 12 in 85% process, using LC-MS for reaction monitoring, are shown
yield. Catalytic hydrogenation of 12 using 5% palladium- in Table 1, and can be summarized as follows: (1) In con-
on-carbon at room temperature in methanol directly pro- trast to the use of 5% palladium-on-carbon, the use of
duced compounds 2 and 3 in only 20 minutes. The free 10% palladium-on-carbon at room temperature led to par-
amine intermediate 1 could not be trapped under these tial reduction of the imine function in the heterocycle.
conditions, demonstrating the high cyclization tendency However, at low temperatures this side reaction was not
of the spacer unit, and thus corroborating our molecular observed, even with an increased amount of 10% palladi-
design.
um-on-carbon. (2) The use of acidic conditions did not
prevent partial fragmentation at room temperature or at 0
°C. When deprotection was complete after 20 minutes, a
small amount of 2 was already formed. (3) Analysis at
meticulously adjusted temperatures proved that the depro-
tection was sufficiently fast at –20 °C. Below –30 °C, no
reaction took place at all, not even with an increased
amount of catalyst. (4) deprotection at –20 °C with 10
mol% of 5% palladium-on-carbon was incomplete after
six hours. During this time, however, a considerable pro-
portion of 1 underwent cyclization to give 2. Increasing
the amount of catalyst fourfold accelerated the reaction
significantly. However, when deprotection was complete
We then evaluated the performance of 1 in buffered aque-
ous media. Rapid fragmentation was by no means guaran-
teed considering the pKa of prolinol (10.09),15
corresponding to only 0.08% of compound 1 in its unpro-
tonated form at pH 7. We therefore intended to isolate 1,
in order to test its behavior at different pH values in buff-
ered aqueous media. To achieve this, deprotection of 12
had to be carried out under conditions that would prevent
the highly favored cyclization. We speculated that depro-
tection under acidic conditions would considerably de-
crease the nucleophilicity of the pyrrolidinyl nitrogen and
thus inhibit fragmentation. Another possible strategy was
Table 1 Optimization of the Reaction Conditions for the Conversion of 12 into 1·HOAc
Entry
Solvent
Catalyst
Temp (°C)
r.t.
Time (min)
Productsa
1
2
3
4
5
6
7
8
9
MeOH
10% Pd/C, (10 mol%)b
5% Pd/C, (10 mol%)
5% Pd/C, (10 mol%)
5% Pd/C, (10 mol%)
5% Pd/C, (10 mol%)
5% Pd/C, (40 mol%)
5% Pd/C, (10 mol%)
5% Pd/C, (40 mol%)
10% Pd/C, (80 mol%)
20
20
2c
MeOH
r.t.
2
MeOH–H2O–AcOH
MeOH–H2O–AcOH
MeOH–H2O–AcOH
MeOH–H2O–AcOH
MeOH–H2O–AcOH
MeOH–H2O–AcOH
MeOH–H2O–AcOH
r.t.
20
1, 2
0
20
1, 2
< –30
< –30
–20
360
360
360
180
90
–
–
1, 12, 2
1, 2
–20
–20
1
a Compounds 2 and 3 are formed simultaneously, but to simplify the table only 2 is listed.
b mol% is the mole percentage of Pd in relation to 12.
c Partial imine reduction in the heterocycle was observed.
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PAPER
A Self-Immolative Spacer–Phenolate Unit
321
after three hours a small quantity of 2 could still be detect- creasing the pH of the initial buffer solution (see above) to
ed. Finally, by doubling the palladium content (i.e., 10% the desired value, a sample was immediately subjected to
Pd/C), deprotection was complete in only 90 minutes fluorescence analysis. Figure 3 shows the three curves
without any detectable trace of 2. Careful evaporation of that were obtained at different pH values. At pH 6, a sig-
the solvent at low temperature furnished the pure hydro- moidal curve was observed that reached a plateau after 30
acetate of 1. This investigation presents a precise correla- minutes. At pH 7 and 8 the initial value did not show any
tion between temperature, amount of catalyst, and evolution over time which can be explained with the im-
reaction time. It may be helpful for other cases where Cbz mediate and complete liberation of fluorophore 3. In all
deprotection is accompanied by side reactions under stan- three cases, the release of the water-insoluble fluorophore
dard conditions.
was characterized by the solution turning cloudy. This be-
havior at physiological pH is in accord with the results
from the LC-MS analyses.
In order to test the cyclization tendency of 1 in aqueous
media, it was dissolved in phosphate buffer at pH 4, where
it was stable. Subsequently, the pH was increased by add-
ing aqueous sodium hydroxide and a sample was taken
from this solution and immediately analyzed by LC-MS
(Figure 2). This way we proved that 1 underwent instan-
taneous cyclization at pH 7 and 8, thus showing the same
behavior as in nonacidified methanol (Scheme 4).
Figure 3 Fluorescence monitoring of the fragmentation of 1 relea-
sing 3 at different pH values
In conclusion, we have conceived a highly preorganized
self-immolative spacer that fragments spontaneously in
methanol and in aqueous media to liberate a fluorescent
precipitate based on the distinctive HPQ system. Applica-
tion of this spacer is, of course, not limited to the genera-
tion of free HPQ, but may well be extended to the
liberation of other leaving groups attached to the car-
bonyl. Its synthesis required us to optimize a sterically
very unfavored case of ether formation. The elevated
yields obtained in this study are so far unique in the liter-
ature and the observations made during reaction optimiza-
tion may turn out to be of use in very different synthetic
contexts. The same holds true for our results in conducting
a catalytic hydrogenation at low temperature in order to
prevent unwanted side reactions of our target compound.
Finally, our observation of the instantaneous fragmenta-
tion of the spacer unit at a wide pH range provide precious
insight into the possibilities in designing new probes for
detection and imaging.
Figure 2 LC-MS monitoring of the fragmentation of 1 at different
pH values; precipitate 3 is filtered off prior to injection into the LC-
MS apparatus, and does therefore not appear in the chromatogram
Reagents and solvents were purchased from Aldrich, Acros, and
Alfa Aesar. HPQ derivative 35 and Cbz-(S)-prolinol11 were pre-
pared as previously described. THF was distilled from Na/ben-
zophenone, and pyridine was dried using KOH prior to distillation.
Column chromatography was performed using Merck silica gel Si
60 (40–63 mm). 1H and 13C NMR spectra were recorded on a Varian
Unity 500 spectrometer (499.83 and 126.7 MHz, respectively), or a
Bruker DPX 200 instrument (200.13 and 50.13 MHz, respectively).
Chemical shifts are referenced from TMS or from solvent referen-
ces.16 HRMS were recorded by the Service Central d’Analyse du
CNRS, Solaize, France. Melting point determinations were carried
out on a Büchi melting point B-540 apparatus and are uncorrected.
For those compounds existing as a mixture of rotamers, the signal
Even at pH 6, 25% of 1 was already cyclized after 5 min-
utes and analysis after 90 minutes showed formation of 2
to be complete. These results are particularly striking in
view of the traces of the pyrrolidine nitrogen actually ex-
isting in its unprotonated form at this pH and, thus, illus-
trate the pronounced preorganization of the spacer.
The fragmentation process was also monitored by fluores-
cence spectroscopy. The water-insoluble HPQ derivative
3 is known to emit a strong fluorescence signal at l = 550
nm with an excitation maximum at l = 370 nm.5 After in-
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322
M. Waibel et al.
PAPER
corresponding to equivalent protons or carbons of different rotam-
ers is separated by ‘and’.
Cyclohexylmethyl Tosylate (9)
Following the typical procedure for 5 using commercial cyclohexyl-
methanol (2.00 g, 17.5 mmol) and TsCl (10.00 g, 52.6 mmol) in an-
hyd pyridine (30 mL) gave 9 as a white solid after recrystallization
(cyclohexane); yield: 3.55 g (76%); mp 32 °C (Lit.12a 30–32 °C).
4-Methoxy-2-(4-oxo-3,4-dihydroquinazolin-2-yl)phenyl
Acetate (7)
Compound 3 (120 mg, 0.45 mmol) was dissolved in anhyd pyridine
(5 mL) and Ac2O (420 mL, 4.5 mmol) was added. The reaction was
refluxed for 2 h then brought to r.t. and quenched by the addition of
H2O (5 mL). The mixture was extracted with Et2O (3 × 5 mL) and
the combined organic extracts were dried (Na2SO4) and the solvent
was removed under vacuum. Compound 7 was obtained as a white
solid after recrystallization (cyclohexane–EtOAc); yield: 119 mg
(85%); mp 194 °C.
13C NMR spectral data were in accord with literature values,12a but
HRMS and 1H NMR data were not reported previously.
1H NMR (500 MHz, CDCl3): d = 7.78 (d, J = 8.2 Hz, 2 H), 7.34 (d,
J = 8.2 Hz, 2 H), 3.81 (d, J = 6.4 Hz, 2 H), 2.45 (s, 3 H), 1.66–1.59
(m, 6 H), 1.26–1.18 (m, 3 H), 0.93–0.86 (m, 2 H).
13C NMR (50 MHz, CDCl3): d = 144.6, 133.4, 129.7, 127.9, 75.3,
37.5, 29.4, 26.2, 25.4, 21.7.
1H NMR (500 MHz, CDCl3): d = 10.40 (br s, 1 H), 8.27 (d, J = 7.7
Hz, 1 H), 8.00 (d, J = 7.7 Hz, 1 H), 7.78–7.72 (m, 2 H), 7.54–7.39
(m, 2 H), 7.24 (d, J = 7.5 Hz, 1 H), 3.82 (s, 3 H), 2.28 (s, 3 H).
HRMS (ESI): m/z [M + H]+ calcd for C14H21O3S: 269.1212; found:
269.1213.
13C NMR (50 MHz, CDCl3): d = 168.8, 162.3, 149.7, 149.1, 148.4,
134.9, 132.3, 130.5, 128.0, 127.2, 126.7, 126.5, 126.0, 123.8, 121.0,
57.5, 21.1.
HRMS (ESI): m/z [M + H]+ calcd for C17H15N2O4: 311.1032; found:
311.1035.
Ethyl 2-(Cyclohexylmethoxy)-2-methylpropanoate (10); Typi-
cal Procedure
NaH (169 mg, 60% in mineral oil, 4.23 mmol) was suspended in
DMF (20 mL) and the mixture was cooled to 0 °C. Commercial al-
cohol 4 (500 mL, 3.67 mmol) was added, and the mixture was
brought up to r.t. and stirred for 30 min. Compound 9 (984 mg, 3.67
mmol) in DMF (5 mL) was added, and the mixture was heated to 80
°C for 2 h. The mixture was cooled to 0 °C, quenched with sat. aq
NH4Cl (20 mL), and extracted with Et2O (3 × 25 mL). The com-
bined organic extracts were washed with brine (2 × 25 mL) and
dried (Na2SO4) and the solvent was removed under vacuum. After
purification by flash chromatography (cyclohexane–EtOAc, 9:1),
10 was obtained as a yellow oil; yield: 761 mg (91%).
1H NMR (500 MHz, CDCl3): d = 4.16 (q, J = 7.1 Hz, 2 H), 3.13 (d,
J = 6.4 Hz, 2 H), 1.78–1.67 (m, 6 H), 1.34 (s, 6 H), 1.28 (t, J = 7.1
Hz, 3 H), 1.25–1.16 (m, 3 H), 0.92–0.85 (m, 2 H).
13C NMR (50 MHz, CDCl3): d = 174.8, 77.1, 70.3, 60.7, 38.4, 30.1,
26.6, 25.8, 24.6, 14.2.
4-Methoxy-2-(4-oxo-3,4-dihydroquinazolin-2-yl)phenyl 2,2-
Dimethylpropanoate (8)
Following the typical procedure for 7 using 3 (120 mg, 0.45 mmol)
with pivalic anhydride (770 mL, 4.5 mmol) in pyridine (5 mL) gave
8 as a white solid after recrystallization (cyclohexane–EtOAc);
yield: 141 mg (89%); mp 162 °C.
1H NMR (500 MHz, CDCl3): d = 9.71 (br s, 1 H), 8.30 (d, J = 7.7
Hz, 1 H), 8.03 (d, J = 7.7 Hz, 1 H), 7.82–7.76 (m, 2 H), 7.53–7.41
(m, 2 H), 7.17 (d, J = 7.5 Hz, 1 H), 3.84 (s, 3 H), 1.34 (s, 9 H).
13C NMR (50 MHz, CDCl3): d = 176.4, 162.5, 150.1, 149.0, 148.7,
134.7, 131.9, 130.4, 127.8, 126.9, 126.8, 126.4, 126.2, 123.3, 120.8,
57.7, 39.1, 26.9.
HRMS (ESI): m/z [M + H]+ calcd for C13H25O3: 229.1805; found:
229.1808.
HRMS (ESI): m/z [M + H]+ calcd for C20H21N2O4: 353.1502; found:
353.1504.
Benzyl (S)-2-{[1-(Ethoxycarbonyl)-1-methylethoxy]meth-
yl}pyrrolidine-1-carboxylate (6)
Following the typical procedure for 10, alcohol 4 (1.03 mL, 7.57
mmol) was deprotonated with NaH (348 mg, 60% in mineral oil,
8.70 mmol) in DMF (40 mL). Reaction with 5 (2.94 g, 7.57 mmol)
in DMF (10 mL) gave 6 as a yellow oil after purification by flash
chromatography (cyclohexane–EtOAc, 7:3); yield: 1.11 g (42%).
Benzyl (S)-2-(Tosyloxymethyl)pyrrolidine-1-carboxylate (5);
Typical Procedure
Cbz-(S)-prolinol11 (11.20 g, 47.66 mmol) was dissolved in anhyd
pyridine (80 mL), and TsCl (27.00 g, 142.11 mmol) was added at 0
°C. The temperature was allowed to rise up to r.t. overnight and was
quenched with H2O (20 mL). EtOAc (80 mL) was added and the
mixture was washed with 1 M HCl (2 × 20 mL), H2O (20 mL), and
brine (20 mL). The organic phase was dried (Na2SO4), and the sol-
vent was removed under vacuum. After flash chromatography (cy-
clohexane–EtOAc, 1:1), pure 5 was obtained as a white solid; yield:
17.06 g (92%); mp 51 °C.
[a]D25 –44.3 (c 1.06, CHCl3).
1H NMR (500 MHz, CDCl3): d = 7.36–7.21 (m, 10 H), 5.18–5.04
(m, 4 H), 4.17–4.07 (m, 4 H), 4.00–3.91 (m, 2 H), 3.59 (dd, J = 8.2,
3.0 Hz, 1 H), 3.50–3.32 (m, 6 H), 3.24–3.20 (m, 1 H), 2.14–2.10 (m,
2 H), 2.00–1.81 (m, 6 H), 1.41 (s, 3 H), 1.37 (s, 3 H), 1.33 (s, 6 H),
1.28 (t, J = 7.1, 3 H) and 1.23 (t, J = 7.1, 3 H).
13C NMR (50 MHz, CDCl3): d = 174.7 and 174.5, 154.9 and 154.3,
137.0 and 136.9, 128.4, 128.0, 127.9, 77.5 and 77.2, 66.7 and 66.5,
65.2 and 64.6, 60.8, 57.6 and 56.9, 47.0 and 46.7, 28.5 and 27.7,
24.9, 24.3, 23.7 and 22.8, 14.2.
[a]D25 –42.6 (c 1.02, CHCl3).
1H NMR spectral data were in accord with the literature values,12b
but HRMS and 13C NMR data were not reported previously.
1H NMR (500 MHz, CDCl3): d = 7.79–7.69 (m, 4 H), 7.37–7.24 (m,
14 H), 5.10–4.97 (m, 4 H), 4.20–3.97 (m, 6 H), 3.42–3.32 (m, 4 H),
2.42 (s, 6 H), 2.00–1.81 (m, 8 H).
13C NMR (50 MHz, CDCl3): d = 154.6 and 154.3, 144.7, 136.5 and
136.3, 132.73 and 132.67, 129.7, 128.4, 127.9, 127.7, 127.6, 69.7
and 69.6, 66.8 and 66.6, 56.0 and 55.3, 46.9 and 46.6, 28.4 and 27.5,
23.7 and 22.7, 21.5.
HRMS (ESI): m/z [M + H]+ calcd for C19H28NO5: 350.1969; found:
350.1974.
Benzyl (S)-2-[(1-Carboxy-1-methylethoxy)methyl]pyrrolidine-
1-carboxylate (11)
Ester 6 (1.15 g, 3.30 mmol) was dissolved in THF–MeOH (30 mL–
40 mL), and aq 1 M LiOH (16 mL) was added. After stirring over-
night at r.t., the pH value was adjusted to 2 by the addition of 1 M
HCl, and the mixture was extracted with Et2O (3 × 30 mL). The
combined organic extracts were dried (Na2SO4) and the solvent was
HRMS (ESI): m/z [M + H]+ calcd for C20H24NO5S: 390.1376;
found: 390.1378.
Synthesis 2009, No. 2, 318–324 © Thieme Stuttgart · New York

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A Self-Immolative Spacer–Phenolate Unit
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removed under vacuum to give pure 11 as a white solid; yield: 1.06
g (quant.); mp 158 °C.
and 56.9, 56.1 and 55.7, 45.9 and 45.5, 27.1 and 26.7, 24.6, 23.6,
22.8 and 22.5.
[a]D25 –44.5 (c 1.04, acetone).
1H NMR (500 MHz, acetone-d6): d = 7.40–7.29 (m, 10 H), 5.19–
5.10 (m, 4 H), 3.98–3.90 (m, 2 H), 3.57 (dd, J = 8.6, 3.8 Hz, 2 H),
3.47–3.32 (m, 6 H), 1.96–1.77 (m, 8 H), 1.37 (s, 6 H), 1.33 (s, 6 H).
13C NMR (50 MHz, acetone-d6): d = 175.8, 155.1 and 154.5, 138.3,
129.1, 128.5, 127.3, 77.8, 66.8, 65.6 and 65.2, 58.3 and 57.7, 47.3
and 47.0, 28.2 and 27.4, 24.6, 24.0, 22.9 and 22.4.
HRMS (ESI): m/z [M + H]+ calcd for C24H28N3O5: 438.2030; found:
438.2032.
3,3-Dimethyltetrahydro-1H-pyrrolo[2,1-c][1,4]oxazin-4(3H)-
one (2)
5% Pd/C (113 mg) was added to a soln of 12 (300 mg, 0.53 mmol)
in MeOH (15 mL). The mixture was stirred under a H2 atmosphere
(1 bar) at r.t. for 20 min, then the catalyst was filtered off and the
filtrate was evaporated to dryness. Purification by flash chromatog-
raphy (CH2Cl2–MeOH, 19:1) gave 2 as a colorless oil; yield: 90 mg
(quant.).
HRMS (ESI): m/z [M + H]+ calcd for C17H24NO5: 322.1655; found:
322.1659.
[a]D25 –47.1 (c 1.00, acetone).
1H NMR (500 MHz, acetone-d6): d = 3.93 (dd, J = 11.2, 3.7 Hz, 1
H), 3.71–3.65 (m, 1 H), 3.47–3.31 (m, 3 H), 1.90–1.77 (m, 3 H),
1.45–1.37 (m, 1 H), 1.29 (s, 6 H).
13C NMR (50 MHz, acetone-d6): d = 176.5, 79.4, 68.7, 57.5, 44.6,
28.0, 25.1, 24.8, 22.7.
Benzyl (S)-2-[(1-{[4-Methoxy-2-(4-oxo-3,4-dihydroquinazolin-
2-yl)phenoxy]carbonyl}-1-methylethoxy)methyl]pyrrolidine-1-
carboxylate (12)
Compound 11 (500 mg, 1.56 mmol) was dissolved in DMF (20
mL), and EDC (419 mg, 2.18 mmol), then 1 M HOBt in NMM (2.34
mL, 2.34 mmol) were added. After 5 min, 3 (418 mg, 1.56 mmol)
in DMF (10 mL) was added and the mixture was stirred at r.t. over-
night. The reaction was quenched with sat. aq NaHCO3 (20 mL) and
extracted with Et2O (3 × 20 mL). The combined organic extracts
were washed with 1 M HCl (20 mL) and brine (2 × 20 mL) and dried
(Na2SO4), and the solvent was evaporated under vacuum. Purifica-
tion by flash chromatography (cyclohexane–EtOAc, 1:1) gave 12 as
a colorless oil; yield: 757 mg (85%).
HRMS (ESI): m/z [M + H]+ calcd for C9H16NO2: 170.1182; found:
170.1186.
LC-MS and Fluorescence Measurements
A 500 mM soln of compound 1·HOAc in 50 mM phosphate buffer
(pH 4) was prepared. Subsequently, the pH was increased to the de-
sired value by adding a few drops of concd aq NaOH. Samples of
the resulting solns were immediately analyzed by LC-MS or fluo-
rescence spectroscopy. Fluorescence measurements were carried
out using a microplate spectrofluorimeter (Spectramax Gemini XS,
Molecular Devices) and black 96-well plates (Nunclone, Nunc
Inc.). Fluorescence was monitored at 550 nm with an excitation
wavelength set to 370 nm. Each point of the curves shown in
Figure 3 is the average value of 5 independently performed experi-
ments. LC-MS analyses were conducted on an Agilent 1100 Series
LC/MSD apparatus using a Synergi Polar-RP 80A (Phenomenex)
column (4 mm, 150 × 4.60 mm). Precipitated 3 was filtered off prior
to injection into the apparatus. MeCN and H2O (containing 0.01%
HCO2H) were used as mobile phase at a flow rate of 0.5 mL/min
(gradient: t = 0 min, 0% MeCN; t = 10 min: 100% MeCN). Mass
analysis was performed using ESI in scan mode.
[a]D25 –45.6 (c 1.03, acetone).
1H NMR (500 MHz, acetone-d6): d = 11.17 (br s, 1 H), 8.20 (d,
J = 7.8 Hz, 2 H), 7.89 (d, J = 7.8 Hz, 2 H), 7.76 (d, J = 7.6 Hz, 2 H),
7.69–7.57 (m, 4 H), 7.53–7.47 (m, 2 H), 7.40–7.26 (m, 12 H), 5.12–
5.01 (m, 4 H), 4.01 (s, 6 H), 3.90–3.81 (m, 2 H), 3.60–3.55 (m, 1 H),
3.49–3.44 (m, 1 H), 3.32–3.18 (m, 6 H), 1.97–1.91 (m, 2 H), 1.81–
1.66 (m, 6 H), 1.45 (s, 6 H), 1.42 (s, 6 H).
13C NMR (50 MHz, acetone-d6): d = 173.0 and 172.8, 162.4, 155.1
and 154.9, 151.7, 149.8, 149.4, 138.3, 135.2, 132.5, 131.2, 129.1,
128.6, 128.5, 127.7 127.6, 127.0, 126.8, 123.9, 123.7, 122.2, 78.2,
66.7, 65.8 and 65.3, 58.1 and 57.5, 56.3, 47.5 and 47.1, 28.4 and
28.1, 25.4, 24.6, 23.1 and 22.8.
HRMS (ESI): m/z [M + H]+ calcd for C32H34N3O7: 572.2398; found:
572.2398.
(S)-2-[(1-{[4-Methoxy-2-(4-oxo-3,4-dihydroquinazolin-2-
yl)phenoxy]carbonyl}-1-methylethoxy)methyl]pyrrolidinium
Acetate (1·HOAc)
Acknowledgment
We thank the French Research Ministry and the C.N.R.S. for finan-
cial support. M.W. and X.Z. acknowledge a European doctoral fel-
lowship and a postdoctoral fellowship (2003 programme Accueil
Jeunes Chercheurs Etrangers), respectively, from the French
Research Ministry.
Compound 12 (200 mg, 0.35 mmol) was dissolved in MeOH (10
mL containing 5 vol% AcOH), and 10% Pd/C (298 mg) was added.
The mixture was cooled to –20 °C and stirred under a H2 atmo-
sphere (1 bar) for 1.5 h. Then the catalyst was filtered off, and the
solvent was removed under vacuum at low temperature using an oil
pump. The residual solid was further dried at r.t. to give the pure
ammonium acetate of 1 as a white powder; yield: 174 mg (quant.);
mp 178 °C.
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Synthesis 2009, No. 2, 318–324 © Thieme Stuttgart · New York